Herpesviruses are etiologic agents of important human diseases including certain cancers. Herpes simplex virus type 1 (HSV-1) is a frequent cause of adult sporadic encephalitis, keratoconjunctivitis and oral mucocutaneous lesions; whereas, herpes simplex virus type 2 (HSV-2) causes genital lesions, meningitis and neonatal infections. HSV infects over 300,000 individuals annually, and they are second only to trauma as a cause of corneal blindness in the U.S.A. Herpes simplex virions acquire viral envelopes via a complicated, sequential de-envelopment and re-envelopment process, in which interactions between tegument proteins and viral glycoproteins embedded in intracellular membranes play important roles. The multi-membrane spanning proteins, UL20p and gK, have been directly implicated in intracellular virion envelopment, since deletion of either gene results in drastic accumulation of unenveloped capsids in the cytoplasm. In addition, both UL20p and gK harbor syncytial mutations suggesting that they are involved in membrane fusion phenomena during virion envelopment and egress, as well as virus-induced cell fusion. We have shown that HSV-1 gK and UL20p are co-dependent for cell-surface expression and TGN localization, and that they physically interact. In addition, we have found that UL20p can physically interact with gB. The proposed investigations focus on the role of herpes simplex virus (HSV) glycoprotein K (gK) and UL20 protein (UL20p) in virus-induced cell fusion and cytoplasmic virion envelopment. The main hypotheses of this grant application are: i) gK and UL20p interact and regulate gB-mediated cell fusion; ii) gK and UL20p function as virion structural components in cytoplasmic virion envelopment by binding to tegument proteins. The specific aims are: I) to characterize UL20p interactions with gK and investigate their coordinate transport to cell-surfaces, endocytosis and TGN co-localization; II) to investigate the role of UL20p interactions with gK and gB on gB-mediated virus-induced cell fusion; III) to investigate the role of UL20p in cytoplasmic virion envelopment. Elucidations of functional interrelationships between gK, UL20p and gB as well as between gK, UL20p and tegument proteins will help define viral protein-protein interactions involved in infectious virus production and may assist in the development of new antiviral strategies.